Plasma Membrane Cholesterol Modulates Cellular Vacuolation Induced by the Helicobacter pylori Vacuolating Cytotoxin

Abstract

The Helicobacter pylori vacuolating cytotoxin (VacA) induces the degenerative vacuolation of mammalian cells both in vitro and in vivo. Here, we demonstrate that plasma membrane cholesterol is essential for vacuolation of mammalian cells by VacA. Vacuole biogenesis in multiple cell lines was completely blocked when cholesterol was extracted selectively from the plasma membrane by using ␤-cyclodextrins. Moreover, increasing plasma membrane cholesterol levels strongly potentiated VacA-induced vacuolation. In contrast, inhibiting de novo biosynthesis of cholesterol with lovastatin or compactin had no detectable effect on vacuolation. While depletion of plasma membrane cholesterol has been shown to interfere with both clathrin-mediated endocytosis and caveola-dependent endocytosis, neither of these two internalization pathways was found to be essential for vacuolation of cells by VacA. Depleting plasma membrane cholesterol attenuated the entry of VacA into HeLa cells. In addition, ␤-cyclodextrin reagents blocked vacuolation of cells that were either preloaded with VacA or had VacA directly expressed within the cytosol. Collectively, our results suggest that plasma membrane cholesterol is important for both the intoxication mechanism of VacA and subsequent vacuole biogenesis.

Different GPI-attachment signals affect the oligomerisation of GPI-anchored proteins and their apical sorting

“…4). As previously shown (Patel et al, 2002), the pre-treatment of MDCK cells with cholesterol (2 mM) in a complex with methyl-β-cyclodextrin (βCD) results in an increase of ~50% in cellular cholesterol levels (see Materials and Methods), without detectable cytotoxicity or morphological and functional changes in the cell monolayer (supplementary material Fig. S2).…”

Cholesterol Depletion ReducesHelicobacter pyloriCagA Translocation and CagA-Induced Responses in AGS Cells

“…Lipid rafts that contain cellular cholesterol have been shown to be utilized by VacA for binding and entry, hence exhibiting intoxication activity (23,29,40,44,46). Wunder et al recently reported that H. pylori absorbs cellular cholesterol and transforms it into glucosylated derivatives to evade phagocytosis during infection (63).…”

“…Several types of evidence suggest that VacA intoxication relies on cellular lipid rafts, which are dynamic microdomains containing cholesterol, sphingolipids, and glycosylphosphatidylinositol-anchored proteins in the exoplasmic leaflet of the lipid bilayer and are implicated in cellular signaling, membrane transport and trafficking, and membrane curvature (for reviews, see references 26 and 51). First, cellular cholesterol is required for VacA cytotoxicity in cultured gastric cells (23,40,46), and VacA migrates with tubulovesicular structures that contain glycosylphosphatidylinositol-anchored proteins to enter cells for its full vacuolating activity (23,29,44). Furthermore, using a cold detergent extraction method, VacA has been isolated primarily on detergent-resistant membranes (DRMs) that are generally considered to be fractions of rafts (23,40,46).…”

“…First, cellular cholesterol is required for VacA cytotoxicity in cultured gastric cells (23,40,46), and VacA migrates with tubulovesicular structures that contain glycosylphosphatidylinositol-anchored proteins to enter cells for its full vacuolating activity (23,29,44). Furthermore, using a cold detergent extraction method, VacA has been isolated primarily on detergent-resistant membranes (DRMs) that are generally considered to be fractions of rafts (23,40,46). It has been noted that cholesterol-rich microdomains are also utilized by other bacterial toxins, including cholera toxin (38) and aerolysin (1), for entry or oligomerization.…”

Effect ofHelicobacter pylori’s vacuolating cytotoxin on the autophagy pathway in gastric epithelial cells

“…For cell intoxication, 10 6 AGS cells were incubated in the presence of activated purified VacA toxin (26.5 x 10 -3 μM) for the time periods indicated in each experiment. 47 VacA mediated with CCMS from wild type or VacA mutant bacteria for 6 hours. Cells were then washed and fixed in a glutaraldehyde based fixative and sections or grids prepared for microscopy (Microscopy Facility, Hospital for Sick Children).…”